We tested the hypothesis that the absence of M3 receptors might protect against different forms of experimentally- and genetically-induced obesity and the associated metabolic deficits. This work was prompted by our previous observation that M3 muscarinic receptor-deficient mice (M3R-/- mice) showed reduced adiposity when maintained on regular mouse chow (Yamada et al., Nature 410, 207-212, 2001). In order to induce obesity, glucose intolerance, and insulin resistance, mice were maintained on a high-fat diet or were treated with gold-thioglucose (GTG) which induces hyperphagia by selectively destroying glucose-receptive neurons in the ventromedial nucleus of the hypothalamus. In addition, we also examined whether the lack of M3 receptors was able to prevent the increase in body weight and the metabolic complications caused by the absence of leptin. To address this issue, we generated and analyzed mutant mice that were deficient in both leptin and M3 receptors (M3R-/- ob/ob mice). For all studies, [unreadable] we used M3R-/- mice that had been backcrossed for ten generations onto the C57BL/6NTac background. [unreadable] In all three experimental models of obesity, M3R-/- mice were largely protected against obesity-associated glucose intolerance, insulin resistance, hyperinsulinemia, and hyperglycemia. Strikingly, in all three mouse models, the lack of M3 receptors was associated with an increase in resting metabolic rate, hyperactivity, and elevated body temperature. It is likely that the resulting increase in energy expenditure represents a major factor contributing to the reduced body weight and adiposity and improved glucose homeostasis observed with the M3R-/- mice. In addition, we demonstrated that M3R-/- mice consumed significantly less food than their wild-type littermates. For example, we found that the lack of M3 receptors was also able to prevent or greatly reduce the hyperphagia caused by GTG administration or by disruption of the leptin gene.[unreadable] We also noted that many of the phenotypic changes exhibited by the M3R-/- mice, including the observed increases in metabolic rate, locomotor activity, and body temperature, are consistent with an elevated tone of the sympathetic nervous system (SNS). In agreement with this notion, we found that urine epinephrine and norepinephrine levels were significantly increased in M3R-/- mice. Although the precise mechanism underlying this increase in sympathetic outflow remains unclear at present, one possibility is that central (e.g. hypothalamic) M3 receptors exert an inhibitory influence on sympathetic outflow in wild-type mice and that the lack of this inhibition in M3R-/- mice triggers the observed elevation in SNS activity. [unreadable] In addition, we found that transcript levels of UCP3, the major UCP expressed in skeletal muscle, were significantly increased in skeletal muscle of M3R-/- mice. Skeletal muscle is known to be the most important contributor to basal metabolic rate and energy expenditure, partially due to proton leak in resting skeletal muscle. One possibility therefore is that increased SNS activity leads to a selective increase in UCP3 expression in skeletal muscle of M3R-/- mice which in turn triggers increased energy expenditure, contributing to the reduction in body weight and obesity associated with the lack of M3 receptors.[unreadable] It has also been proposed that UCP3 may act as a mitochondrial fatty acid efflux protein, thus stimulating fatty acid oxidation. In agreement with this notion, we found that M3R-/- mice displayed a pronounced increase in the rate of fatty acid oxidation in vivo. This elevated fatty acid oxidation rate could be greatly reduced by treatment of M3R-/- mice with the alpha-receptor blocker phentolamine, indicating that increased sympathetic tone makes a major contribution to this phenotype. [unreadable] Quantitative RT-PCR studies showed that the expression of PDK4 (pyruvate dehydrogenase kinase 4) was also upregulated in skeletal muscle from M3R-/- mice. PDK4 inactivates pyruvate dehydrogenase via phosphorylation, thus conserving 3-C compounds under conditions when glucose availability is limited and stimulating fatty acid oxidation for the generation of ATP. Because of the large contribution of skeletal muscle to whole body glucose disposal, suppression of pyruvate dehydrogenase by PDK4 in skeletal muscle is likely to be of particular relevance for glucose homeostasis. Our data are therefore consistent with the concept that the lack of M3 receptors leads to a relative shortage of glucose, resulting in increased expression of PDK4 in skeletal muscle, reduced oxidative breakdown of glucose, and increased mitochondrial fatty acid oxidation to ensure proper ATP production.[unreadable] Under all experimental conditions used, the absence of M3 receptors was associated with a striking increase in insulin sensitivity. Euglycemic-hyperinsulinemic clamp studies demonstrated that deletion of the M3 receptor gene led to a pronounced stimulation of glucose uptake in skeletal muscle and white adipose tissue (WAT). Radioligand binding studies indicated that skeletal muscle and WAT preparations from wild-type mice did not express detectable levels of M3 or other muscarinic receptors, excluding the possibility that the increase in insulin sensitivity displayed by the M3R-/- mice is due to the lack of M3 receptors in these tissues. Since M3R-/- ob/ob mutant mice showed only a rather small reduction in adiposity (as compared to ob/ob mice), but displayed greatly improved glucose tolerance and insulin sensitivity, it is likely that factors unrelated to overall adiposity, probably involving central cholinergic pathways, also contribute to the beneficial metabolic effects associated with the absence of M3 receptors.[unreadable] In summary, we showed that the lack of M3 receptors has pronounced effects on energy and glucose homeostasis in mice. M3R-/- mice are largely protected against the detrimental metabolic deficits triggered by a high-fat diet, chemical disruption of ventromedial hypothalamic neurons by GTG, and genetic disruption of the leptin gene. The M3 receptor may therefore represent a potential pharmacologic target for the treatment of obesity and associated metabolic disorders.